Echinococcosis, caused by cestodes of the genus Echinococcus, poses significant public health and veterinary concerns globally. In Armenia, cystic echinococcosis (CE) is well-documented in livestock and humans, while alveolar echinococcosis (AE) has long been considered non-endemic. However, a recent retrospective study identified human AE cases, suggesting an underestimation of the parasite’s presence. To address knowledge gaps, a pilot survey was conducted to identify Echinococcus species and other taeniids in free-roaming dogs and wild carnivores in Armenia. Fecal samples (n = 112) were opportunistically collected from eight wild carnivore species and stray dogs across six Armenian provinces between 2017 and 2018. Samples were analysed for taeniid eggs using flotation and molecular techniques. Echinococcus multilocularis was identified in a free-roaming dog, marking the first confirmed detection of the parasite in a definitive host in Armenia. Additionally, E. canadensis G6/7 and E. ortleppi were detected in a wolf, while E. canadensis G6/7, Taenia hydatigena, and T. laticollis were found in a lynx. These findings indicate the involvement of both domestic and wild carnivores in the transmission cycles of Echinococcus species and suggest the presents of a potential sylvatic cycle involving E. canadensis G6/7 and E. ortleppi. The presence of E. multilocularis in a dog underscores the risk of human infection, necessitating further epidemiological studies. This study provides initial insights into the epidemiology of Echinococcus in Armenia and highlights the need for continued surveillance to assess public health risks.

Male genital schistosomiasis (MGS), a gender-specific manifestation of urogenital schistosomiasis and neglected tropical disease, typically results from the entrapment of Schistosoma haematobium eggs within the male genital tract. Across the world, there are no current and accurate estimates of the burden of MGS, due to disease underreporting primarily from diagnostic challenges and a lack of general awareness within the health system. Diagnostic methods for MGS are extremely limited. Conventionally, semen microscopy for Schistosoma ova is used though this technique suffers from low sensitivity and lacks protocol standardization. The introduction of molecular diagnostics, such as polymerase chain reaction (PCR), has partly helped overcome this challenge of low sensitivity, though may not be suitable for use in resource-constrained settings. To address these challenges, in this review, we propose a two-step diagnostic algorithm for MGS in accordance with recent WHO guidelines, consisting of a high sensitivity serological test followed by a high specificity test (microscopy or molecular assay, dependent on setting). Further investigation is required into standardization of sample collection, processing, storage, and analysis in order to identify an evidence-based optimal diagnostic pipeline. New diagnostic tools are needed such as isothermal molecular assays, alongside optimization for semen analysis, which may alleviate barriers to diagnosis and present opportunities for integration with other sexual and reproductive health screening. These areas of future investigation underpin the development of a suitable diagnostic pipeline, as the continued neglect of MGS and its underdiagnosis presents a threat to the goal of elimination of schistosomiasis as a public health problem.

Gastrointestinal (GI) nematode infections represent a significant health burden globally, affecting both humans and livestock. Traditional in vitro models to study host–parasite interactions, such as immortalized cell lines, have limitations that hinder the full understanding of these complex relationships. Organoid technology has emerged as a promising alternative, offering a physiologically relevant platform to study host–nematode interactions in vitro. Organoids are three-dimensional structures comprising differentiated cell types that recapitulate features of the corresponding organ. Technological advances for growing, maintaining and manipulating organoids have increased their applications to model infections, inflammation and cancer. This review discusses recent work using GI organoids to advance understanding of nematode–host interactions and modulation of GI epithelial cells. Additionally, we review studies that co-cultured GI organoids with innate lymphoid cells to study epithelial-immune cell cross-talk in the context of nematode infection. By bridging the gap between reductionist cell culture systems and whole-organism studies, organoids offer a powerful platform for investigating complex host–nematode interactions, and for developing and screening novel therapeutics.

Acanthocephalans, which are in the family Polymorphidae, are a globally distributed group of endoparasites whose adults reside in the intestines of fish-eating birds, waterfowl and marine mammals. Adults of Polymorphus brevis and Pseudocorynosoma constrictum are endoparasites of fish-eating birds (Ardeids) and waterfowl (Anatidae), respectively, and are considered one of the most abundant and widely distributed species of polymorphids in freshwater systems from the Nearctic and Neotropical regions of Mexico and the USA. In the present study, sequences of cytochrome c oxidase subunit 1 (cox1) from mitochondrial DNA were generated from 67 specimens of P. brevis and 32 of Ps. constrictum from 12 localities on 6 biogeographic provinces in Mexico (the Trans-Mexican Volcanic Belt, Pacific Lowlands, Veracruzan, Californian, Sierra Madre Occidental, and Sonoran), plus the Temperate Prairies biogeographical province in the USA. The phylogeographic analyses indicated that the populations of both species lacked phylogeographic structure and exhibited high haplotype diversity, low nucleotide diversity and low Fst values among the biogeographic provinces; in combination with negative values in the neutrality test, these findings suggest that the populations of both species of acanthocephalan are undergoing expansion. The current evidence indicates that the biology of the definitive hosts, in combination with their migration patterns, could play a key role in shaping the distribution of haplotypes and the population genetic structure of the studied 2 acanthocephalan species.

It is often postulated that natural systems are expected to suffer an increasing risk of infectious disease outbreaks as climate change accelerates. In the northern Argentine Sea, the rise of ocean temperature has produced a tropicalization of demersal megafauna since 2013. This rapidly warming hotspot provides an excellent model to test whether fish parasites have increased, declined, or remained stable in the region. Cardiocephaloides physalis a parasite of penguins Spheniscus magellanicus as adult and suspected to parasitize anchovies Engraulis anchoita as larvae is here used to compare their occurrence and abundance between samples composed by 1752 fish of variable age caught at different latitudes during 1993–1995 and 2022 and between 20 juvenile birds and literature data. In the present work, the identity of metacercariae as C. physalis is confirmed genetically, as well as a net decline of population parameters of the parasite to its effective disappearance in anchovies from northern areas and to extremely low levels in fish from southern regions and penguins. After analysing possible causes for such changes in a scenario of rapid regional tropicalization, a direct effect of increasing temperature on parasites arose as the main causal candidate for the observed decline in their populations over the last decades. Beyond the biological and ecological consequences of global change on them, parasites offer excellent systems for measuring and monitoring such effects. The almost local extinction of C. physalis in a marine hotspot of global warming seems to be one of the first examples of such processes.

Transposable elements (TEs) have the ability to move and amplify inside the host genome, making them a pivotal source of genome plasticity. Presently, only 4 TE clades (all classified as Class I retrotransposons) have been identified in trypanosomatids. We predicted repeat content and manually curated TEs across the genomes of 57 trypanosomatids, shedding light on their proportions, diversity and dynamics. Our analysis yielded 214 TE consensus sequence models across the dataset, with abundance ranging from 0.1% to 7.2%. We found evidence of recent transposon activity in most species, with notable bursts in the Vickermania, Lafontella, Porcisia and Angomonas spp., along with Leishmania (Mundinia) chancei, L. (M.) orientalis and L. (M.) procaviensis. We confirmed that the 4 TE clades have colonized virtually all lineages of trypanosomatids, potentially playing a role in shaping their genome architecture. The effort of this work culminated in the establishment of the Trypanosomatid TE Database 1.0, a resource designed to standardize the TE annotation process that can serve as a foundation for future studies on trypanosomatid TEs.

The control of African animal trypanosomosis (AAT) relies on accurate diagnostic tools. Serological diagnosis using ELISA is well-suited for surveillance due to its high-throughput capacity, low cost, and adaptability to rapid formats. However, the WOAH-recommended antibody ELISA for AAT, based on trypanosome lysates purified from rodent blood, can lack specificity and presents standardization challenges as well as ethical concerns. Recombinant proteins offer a solution to standardization, often improving specificity, though potentially at the expense of sensitivity. Combining multiple recombinant proteins can enhance sensitivity while maintaining specificity. Therefore, this study developed chimeric proteins for serological diagnosis of AAT, composed of highly immunoreactive regions from multiple known antigens using genetic engineering. Following an inventory of immunodominant antigens, we selected candidates and, using bioinformatics, designed five chimeric constructs in silico: three species-specific and two pan-trypanosome. The coding sequences for these chimeras were synthesized, cloned into expression vectors, and expressed in Escherichia coli. Purification was achieved through a series of chromatographic steps, including Ni-NTA affinity chromatography, Q Sepharose ion-exchange chromatography, and Sephadex 200 size-exclusion chromatography. Preliminary assessment of their reactivity with sera from cattle experimentally infected with Trypanosoma vivax, T. congolense or T. brucei yielded promising results. Longitudinal analysis comparing their reactivity with trypanosome lysates revealed that those specific to T. congolense, and T. vivax, as well as one pan-trypanosome, can discriminate pre- and post-infection sera. These observations confirm the potential of our chimeric constructs. Future work will involve evaluating these chimeras against a broader panel of sera.

Gastrointestinal infections constitute a significant global health concern, particularly in tropical and subtropical regions, caused by various pathogens. Among these, Cryptosporidium spp. and Giardia duodenalis are noteworthy due to their zoonotic potential. In Algeria, molecular epidemiological data on cryptosporidiosis and giardiasis are limited. To fill this gap, the present study aimed to examine the transmission dynamics of Cryptosporidium spp., and Giardia duodenalis in various households. A total of 216 samples were collected from the rural Guelma and Souk Ahras provinces, located in the eastern part of Algeria. These included human and animal faeces, as well as water and soil samples. DNA was extracted, followed by nested PCR targeting the SSU rRNA gene to detect Cryptosporidium spp., while the gp60 gene was amplified for subtyping. Detection of G. duodenalis was performed by qPCR targeting the SSU rRNA gene, followed by amplification of tpi, bg and gdh genes for genotyping and subtyping. Several Cryptosporidium species, including C. bovis, C. ryanae, C. andersoni and C. parvum, were identified in human, animal and environmental samples. The zoonotic C. parvum subtype IIaA17G2R1 was detected in human, animal and soil samples. Giardia duodenalis assemblage B was detected in a human sample, while assemblage E was found in cattle and sheep. The current investigation underscores the importance of the One Health approach in addressing issues related to intestinal parasites, highlighting the need for improved surveillance and control measures in rural settings.

After its near eradication in the 1940s, the common bed bug (Cimex lectularius) experienced a global resurgence. Within a few years after, some populations displayed insecticide resistance. Two distinct lineages of bed bugs were identified, each associated with humans and bats, respectively. A strong genetic differentiation was identified between bugs from human and bat sites across Europe. This raises the question of whether the same pattern is found at a local scale. Moreover, because long-distance dispersal of bed bugs is essentially human-mediated, we investigated the spread of bed bugs within and among sites. Using mitochondrial (cytochrome oxidase unit I (COI) and 16S rRNA genes) and nuclear (10 microsatellite loci) markers, we compared the genetic composition of human- and bat-associated bed bugs from western Switzerland. We first conducted a median-joining analysis and compared it to European sequences to detect local-scale host-specific separation of haplotypes. We estimated levels of genetic diversity and structure between and within the two host-associated bed bugs. Our results reveal two genetic clusters associated with bats and humans and a strong structure among human sites (FSC = 0·579). An analysis of knock-down insecticide resistance gene variants (V419L, L925I, I936F) shows that bed bugs infecting humans in western Switzerland carry insecticide resistance (99%) whereas bed bugs infecting bats do not (0%). Our results show that at the scale of western Switzerland, bed bugs are structured by host association, thus supporting the hypothesis of host specialization in the common bed bugs. Moreover, human-associated bugs might have settled from multiple colonization events and/or undergone bottlenecks.

Avian Plasmodium parasites can be pathogenic to their vertebrate hosts. Although cases of anaemia are frequently reported in parasitized birds, the potential damage caused by the parasite during the exoerythrocytic reproduction phase remains poorly investigated. Here, we report 2 individuals of red-legged seriemas (Cariama cristata) infected with 2 different lineages of Plasmodium huffi, one of them exhibiting potential malarial-compatible tissue lesions in the spleen, liver, brain and lungs, alongside molecular confirmation of parasite presence in the spleen. Previously classified as specific to birds from the order Piciformes, this parasite has shown different associated lineages amplified across diverse host orders in South America (Anseriformes, Charadriiformes, Columbiformes, Galliformes, Pelecaniformes and Passeriformes). Those infections, however, were defined as abortive due to the absence of gametocytes visualized in blood smear slides. Herein, we confirm P. huffi as a generalist parasite based on the first morphological characterization in the peripheral blood of a bird outside the Piciformes order. This is also the first morphological and molecular description of a Plasmodium species in Cariamiformes. In addition to the morphological analyses, we have also proposed a novel phylogenetic hypothesis based on the partial cytb gene and the near-complete mitochondrial genome of this parasite. Our findings support that the division of the genus Plasmodium into subgenera is not monophyletic, as P. (Huffia) huffi and its associated lineages cluster more closely with Plasmodium (Haemamoeba) gallinaceum than with Plasmodium (Huffia) elongatum.

Comparative histopathological and ultrastructural investigations were performed on the livers of 2 fish species, namely, flounder (Platichthys flesus (L.)) naturally infected with the nematode Anisakis simplex (s.l.) (Rudolphi, 1809) larvae (L3) and tuvira (Gymnotus inaequilabiatus) (Valenciennes, 1839) harbouring the nematode Brevimulticaecum sp. (L3) (Shikhobalova and Mozgovoi, 1952). The intensity of infection by A. simplex (s.l.) larvae (L3) in flounders ranged from 3 to 10 parasites per organ. The worms were encapsulated by the peritoneal visceral serosa on the external surface of the liver. Infected P. flesus livers showed hepatocyte cytoplasmic rarefaction and cell swelling. A few immune cell types, such as macrophages, limited numbers of mast cells (MCs), lymphocytes and some epithelioid cells, were observed within the granuloma. The intensity of infection by Brevimulticaecum sp. (L3) in G. inaequilabiatus ranged from 4 to over 340 larvae per organ, and the nematode larvae were encircled by round-to-oval granulomas. Each granuloma possessed 3 concentric layers of cells and tissue: an inner layer in close proximity to the Brevimulticaecum sp. (L3) cuticle, formed by densely packed layers of epithelioid cells showing several desmosomes between each other; a middle layer of numerous MCs entrapped in a thin fibroblast-connective mesh; and an outer layer of fibrous connective tissue with thin, elongated fibroblasts. High numbers of macrophages and macrophage aggregates were scattered within the granuloma. This is the first study to compare the cellular nature of granulomas and the immune responses in the livers of paratenic fish hosts of 2 nematode species.

MicroRNAs (miRNAs), derived from extracellular vesicles (EVs) are circulating intercellular communicators which influence pathogenesis and could be used as potential diagnostic markers. In this study, plasma-derived EVs from Plasmodium knowlesi-infected patients (n = 13) and healthy individuals (n = 10) were isolated using size exclusion chromatography and ultracentrifugation. The presence of EVs was confirmed by transmission electron microscopy and Western immunoblotting, and quantified by nanoparticle tracking analysis. The EVs isolated from patients exhibited a larger size, accompanied by an elevated concentration of EVs. The relative expression levels of 8 human miRNAs were quantified using reverse transcriptase quantitative polymerase chain reaction. Compared to uninfected groups, hsa-miR-223-5p (P-value = 0.0002) and hsa-miR-486-5p (P-value = 0.025) were upregulated in P. knowlesi-infected patients. Bioinformatic analysis revealed that these miRNAs are predicted to target both human host and parasite genes, and they were found to be enriched in various malaria-related pathways. The areas under the receiver operating characteristic curve of hsa-miR-223-5p and hsa-miR-486-5p were 0.9154 and 0.8231, respectively, suggesting the potential of EV-miRNAs as diagnostic markers. Results revealed that EV-miRNAs may play a significant role in the progression of P. knowlesi infection. Further investigations should explore their potential impact on gene expression regulation as diagnostic biomarkers or targets for therapeutic interventions.

Ochoterenella is a large group of filarial parasites of anurans distributed throughout Central and South America. In the present study, we describe a new species of Ochoterenella parasitizing 2 frogs, Boana geographica and Boana multifasciata, from different localities in the Brazilian Amazon. The main morphological traits that differ Ochoterenella casiraghii n. sp. from its congeners are the smaller body size, a shorter cephalic plate, smaller parastomal structures, and the small, short and rounded cuticular bosses on the body of both sexes. The females have a shorter ovejector, and the number of caudal papillae distinguishes males. Pairwise sequence comparisons of the new species reveal a high level of divergence from Ochoterenella spp. Our phylogenetic analyses, based on cox1 and concatenated partial mitochondrial genes, support the monophyly of all subfamilies and genera examined herein. The new species represents the 17th in the Ochoterenella genus and a new parasite record for both anuran species. We provide the first ultrastructural description of the species in the genus and establish the phylogenetic relationships of the new species among parasites of amphibians and reptiles from the Onchocercidae.

Cestodiases, like echinococcoses and cysticercoses, represent a global health problem. Currently available anthelmintics, as benzimidazoles and praziquantel, have limited effectiveness against these cestodiases, creating a demand for the identification of new and more effective drugs. Here, the potential of statins (simvastatin and fluvastatin), for repositioning as novel anthelmintic is explored. Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a main enzyme in the mevalonate pathway, which is vital for the synthesis of non-steroidal isoprenoids and the maintenance of normal cell functioning. A survey for HMG-CoA reductase encoding genes showed that they are present in a single copy in the genomes of parasitic helminths and their mammal hosts. Sequence alignments and phylogenetic analyses showed 20–95% overall identities among ortholog HMG-CoA reductases, with special conservation of their catalytic domains. The HMG-CoA reductase 3D-structure was predicted for orthologs from 3 cestodes of medical importance (Echinococcus multilocularis, Echinococcus granulosus sensu lato, and Taenia solium), and from a model cestode species (Mesocestoides corti). Molecular docking between cestode HMG-CoA reductase orthologs with simvastatin demonstrated that the Arg, Ser, Lys, and Glu residues in conserved positions of the active site interact with this drug, similarly to the interactions predicted for the human reference ortholog enzyme. Furthermore, in vitro assays demonstrated that simvastatin produced a significant reduction of M. corti viability, being able to reduce 100% of parasite viability at 150 μm. Fluvastatin was also assessed showing a lower, although significant anthelmintic effect. The predicted overall structures and interactions together with in vitro assays suggest that cestodes HMG-CoA reductases are inhibited by simvastatin, being a potential therapeutic target for the repurposing of simvastatin as anthelmintic drug. Furthermore, these results pave the way for the in vivo evaluation of the potential effects of simvastatin on cestode larvae.

Accurate characterization of helminth communities in amphibian hosts is essential for understanding host-parasite dynamics in changing environments. This study presents an integrative parasitological survey of Bufotes viridis populations in eastern Slovakia, using both morphological and molecular methods. A total of 61 road-killed individuals collected across 13 localities were examined for helminth presence. Only nematodes were detected, encompassing 3 families, Rhabdiasidae, Molineidae and Cosmocercidae. Four nematode species were recorded, Rhabdias rubrovenosa, Oswaldocruzia filiformis, O. ukrainae and Aplectana linstowi, and a further unidentified Cosmocerca species. Notably, R. rubrovenosa seems to be a new or previously misidentified helminth species found in Slovakia. Cosmocercidae represented the most abundant family, while Molineidae occurred scarcely. Each species was characterized genetically – for the members of Rhabdiasidae and Cosmocercidae, partial 18S rDNA, complete ITS1, complete 5.8S rRNA, complete ITS2 and partial 28S rDNA sequences were amplified, whereas for representatives of Molineidae, partial COI sequences were obtained. These results underscore the utility of combining molecular and morphological tools in helminth biodiversity studies and provide updated baseline data on nematode infections in B. viridis within an anthropogenically influenced landscape. Despite visual patterns indicating differences in the community compositions of nematode families between urban and rural localities, multivariate analyses testing revealed no significant differences.

Host–parasite adaptation is crucial for evolutionary success of a parasite. The nematode Angiostrongylus cantonensis has a heterogenic life cycle involving molluscs as intermediate hosts and rats as definitive hosts. Several mollusc species are susceptible hosts of A. cantonensis, allowing the development of first-stage larvae (L1) into third-stage larvae (L3). Changes in the metabolism of infected molluscs have been demonstrated, disturbing regular routes and inducing host defence mechanisms. This study aimed to identify changes in the proteomic content of Phyllocaulis spp. mucus during A. cantonensis infection. Proteins were extracted from the mucus samples of infected and non-infected slugs and identified using liquid chromatography–tandem mass spectrometry. We found 26 up-regulated and 15 down-regulated proteins in infected slugs compared to non-infected slugs. Protein profiles are promising markers of parasite infection, and a better understanding of proteomic profiles during infection may help inform in vivo studies and promote new tools for the non-invasive identification of infected hosts.

The nutria was introduced to Europe from South America and kept for the fur industry. This semiaquatic rodent became a well-established species in the Czech Republic; however, it still poses a significant threat to the native fauna, not only as a natural competitor but also as a vector of non-indigenous parasites. Our research aimed to investigate the diversity of endoparasitic helminths in nutria, with a particular focus on assessing the risk posed by helminth species with zoonotic potential. A total of 46 nutria cadavers were collected at 8 locations in the Morava River basin and examined using standard parasitological post-mortem procedures. Additionally, coprological and molecular methods were used to identify the parasites. The presence of 6 helminth species was revealed. The highest prevalence was observed for Strongyloides myopotami (78.3%) and Trichuris myocastoris (37.0%), both of which are host-specific nematodes of nutria. Only 2 trematode taxa were recorded (Echinostoma sp. and a representative of the family Psilostomidae). The presence of alveolar hydatid cysts of Echinococcus multilocularis in the livers of 5 nutria specimens was also recorded. Herein, we provide novel molecular data for each parasite species collected, which is valuable for future phylogenetic analyses. Our findings also demonstrate that nutria in the Czech Republic serve as a carrier of helminths with zoonotic potential, particularly E. multilocularis and S. myopotami. Although the nutria is a relatively new species in local fauna, its synanthropic behaviour raises concerns about potential threats to human health, underscoring the importance of exercising caution when handling these animals.

The migratory phase is a critical time for Fasciola hepatica as it must locate, penetrate and migrate through the alimentary tract to the liver parenchyma whilst under attack from the host immune response. Here, scanning and transmission electron microscopy were used to monitor the in vitro effects of sera (with, and without, complement depletion) on F. hepatica newly excysted juveniles (NEJs) and flukes recovered at 7, 35, 70 and 98 days post infection (dpi) from the liver and bile ducts of rats. Test sera were from these F. hepatica-infected rats. A F. hepatica NEJ-specific rabbit antiserum was also used. All fluke stages demonstrated release of the tegumental glycocalyx and microvesicles and intense activity within the tegumental syncytium characterized by eccrine secretion of T-0/T-1/T-2 secretory bodies with subsequent microvillar formation and shedding of microvesicles from the apical plasma membrane. Exposure of both NEJs and 35 dpi flukes to 35 and 70 dpi rat sera produced significant amounts of eccrine-derived secretory material and putative attached immunocomplex. Rabbit anti-F. hepatica NEJ-specific antiserum produced similar responses at the NEJ tegument, including binding of putative immunocomplex to the surface, but with additional blistering of some regions of the apical plasma membrane. Our data suggest that immune sera stimulates multiple interrelated secretory mechanisms to maintain the integrity of the tegumental barrier in response to immune attack. Concurrent release of microvesicles may also serve to both divert the immune response away from the fluke itself and permit delivery of immunomodulatory cargo to immune effector cells.

Elevated levels of IL-6 in plasma are associated with the severity of visceral leishmaniasis (VL). The clinical manifestations of VL vary among patients, influenced by host factors and the virulence of the Leishmania infantum parasite. Considering that severe VL may result from an exaggerated inflammatory response, this study investigated whether IL-6 could serve as a biomarker to identify pro-inflammatory virulence factors. We conducted a genome-wide association study (GWAS) analysis on L. infantum isolates from patients with VL, whose IL-6 concentrations were measured. The analysis revealed that the relationship between IL-6 levels and clinical outcomes (survival vs mortality) had an area under the curve (AUC) of 0.67 (95% CI 0.52–0.81). A cut-off of 391.7 pg mL−1 for IL-6 was established to conduct a logistic regression analysis. We identified 10 029 single nucleotide variants (SNVs) across 62 genomes, resulting in 6,948 SNVs after filtering, of which 6,341 are located in protein-coding regions. The association analysis with PLINK identified 722 variants, of which 35 showed significant associations, with odds ratios ≥3.3, primarily in coding regions. These findings demonstrate that IL-6 levels tended to be associated with the fatal outcome of VL and highlight 35 novel genetic variants that could serve as potential biomarkers for prognosis. Further research into the biological role of these variants may lead to new therapeutic targets and improve the clinical management of VL, especially in identifying high-risk patients.